This R21 exploratory/developmental project in translational research aims to test the small-molecule necrostatin-1 (Nec-1) as a potential neuroprotective agent for the treatment of amyotrophic lateral sclerosis (ALS), an incurable fatal paralytic disorder. Mutations in superoxide dismutase-1 (SOD1) cause familial ALS and studies in chimeric or conditional mutant mice indicate that non-neuronal cells play an important role in mutant SOD1-related neurodegeneration. We and others find that wild-type primary spinal motor neurons (MNs) are selectively killed by mutant SOD1-expressing astrocytes or their conditioned medium by programmed cell death. Now, we show that Nec-1, an allosteric inhibitor of the kinase receptor interacting protein-1 (RIP1), protects against this mutant astrocyte-induced MN death. Thus, to define the cellular target of Nec- 1, specific aim (SA)-1 will determine whether the protection afforded by Nec-1 in our MN/astrocyte co-culture model system of ALS is linked to an inhibition of RIP1 within MNs and/or within astrocytes. Whether Nec-1 protects MN cell bodies as potently as their nerve processes will also be examined. We also find, by in silico modeling, that Nec-1 is predicted to cross the blood-brain barrier raising the possibility that Nec-1 may be usable in vivo as a therapeutic agent for ALS. Thus, in our initial steps toward testing this possibility, SA-2 will assess Nec-1 and its inactive structural analogue Nec-1ia solubility and stability in suitable vehicles for chronic in vivo use, and their central nervous system penetration, pharmacokinetics, routes of administration and tolerability in wild- type mice. Then, based on the optimal conditions of Nec-1 administration defined in SA-2, SA-3 will determine the neuroprotective potency of Nec-1 in the transgenic mutant SOD1 mouse model of ALS using a comprehensive set of behavioral and morphological investigations. We anticipate that, by the end of this project, the preclinical suitability and effectiveness of Nec-1 will have been evaluated, which may lead into a therapeutic development project for ALS. The latter will include preclinical regulatory studies as part of the prerequisites for future clinical trials. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an incurable fatal paralytic disease in which inflammation is an increasingly recognized contributor to the disease process. We have found in a dish that a small molecule called necrostatin blocks the deleterious effects of specific inflammatory cells on the nerve cells responsible for ALS paralysis. Herein, we propose to determine the suitability of using this small molecule is a living organism and to demonstrate whether it is protective in an experimental model of ALS.